Bile acid sulfate sulfatase, process for its preparation and method for assaying bile acid

ABSTRACT

The invention provides a novel bile acid sulfate sulfatase, a process for its preparation, and a method of assaying bile acid 3α-sulfates and total bile acids using the bile acid sulfatase.

This is a division of application Ser. No. 07/548,932, filed July 26,1990.

FIELD OF THE INVENTION

The present invention relates to bile acid sulfate sulfatase, itspreparation and methods of quantitatively determining bile acid.

The enzyme of the invention is a useful novel enzyme for clinicalexaminations, and is useful for the determination of sulfated bile acidssuch as bile acids in which the OH group at 3-position is sulfated(hereinafter referred to as bile acid 3α-sulfate), and for thedetermination of bile acid in blood or urine.

PRIOR ART

It is well known that the bile acid in blood or urine markedly increasesdue to hepatobiliary diseases. It is therefore an important item todetermine the bile acid in blood or urine for evaluating the hepaticfunction in clinical examinations. Conventional method of determiningbile acid adopts the enzymatic analysis method wherein 3α-hydroxysteroiddehydrogenase is employed. Said dehydrogenase oxidizes bile acidswherein the 3-position OH group is in α-configuration (3α-hydroxy bileacid) to 3-oxo bile acids, and concurrently reduces the coenzyme β-NADto NADH. By determining the NADH, the 3α-hydroxy bile acids aredetermined. In the conventional clinical examination, the quantity of3α-hydroxy bile acids is regarded as the total amount of bile acids.

However, bile acids in blood are present in part as sulfated bile acidswhich have been sulfated, and the hydrophilic property is enhanced bythe sulfation and the excretion thereof is facilitated, so that theratio of sulfated bile acids, in particular bile acid 3α-sulfates, inurine is extremely increased. The dehydrogenase in the prior art methodacts only on the 3α-hydroxy bile acids, and hence in the prior artmethod it is impossible to determine the bile acid 3α-sulfates whereinthe 3α-hydroxyl group has been sulfated. In clinical examinations, it isnecessary to determine the total bile acid 3α-sulfates or the total bileacids including it in urine or blood, but bile acid sulfatase capable ofspecifically hydrolyzing the sulfate ester moiety of the bile acid3α-sulfates is not known yet.

Under the circumstances, in order to determine the bile acid3α-sulfates, a sample therefor must be subjected to a columnchromatography to separate bile acid 3α-sulfates, and then the sulfatemoiety of the bile acid 3α-sulfates must be chemically hydrolyzed bysolvolysis. Such process is, however, very cumbersome and cannot be usedeasily in the daily clinical examinations.

DISCLOSURE OF THE INVENTION

It is an object of the invention to provide a novel bile acid sulfatesulfatase capable of specifically hydrolyzing the sulfate ester moietyof bile acid 3α-sulfates.

It is another object of the invention to provide a method for thepreparation of such bile acid sulfate sulfatase.

It is another object of the invention to provide a method capable ofeasily determining the total bile acid 3α-sulfates in blood or urinewhich were impossible to measure in the conventional enzymatic method.

It is a further object of the invention to provide a method capable ofeasily determining the total bile acids, including the bile acid3α-sulfates, in blood or urine.

Other objects and features of the invention will become apparent fromthe following description.

The present invention provides a bile acid sulfate sulfatase, itspreparation, and methods of determining bile acids, as defined in 1 to 4below.

1. A bile acid sulfate sulfatase having the following properties:

(a) Action: Acting on 3α-sulfates of bile acids to hydrolyze the sulfateester moiety thereof and to invert the bonding configuration of theresulting OH group from α-configuration to β-configuration, therebyproducing 3β-hydroxy bile acids;

(b) Substrate specificity: Acting on 3α-sulfates of nonconjugate bileacids, and 3α-sulfates each of glycine-conjugated and taurine-conjugatedbile acids; and

(c) Optimum pH range: pH 8.5±0.5.

2. A method for the preparation of a bile acid sulfate sulfatasecomprising culturing Pseudomonas testosteroni in a culture mediumcontaining bile acid, and recovering the bile acid sulfate sulfatase ofclaim 1 from the resulting culture.

3. A method of determining 3α-sulfates of bile acids comprising causinga bile acid sulfate sulfatase and β-hydroxysteroid dehydrogenase to acton a sample containing 3α-sulfates of bile acids in the presence ofβ-NAD, and determining the produced NADH.

4. A method of determining total bile acid comprising causing a bileacid sulfate sulfatase, β-hydroxysteroid dehydrogenase and3α-hydroxysteroid dehydrogenase to act on a sample containing3α-sulfates of bile acids and other bile acid(s) in the presence ofβ-NAD, and determining the produced NADH.

The present inventors, as a result of intensive researches, succeeded inobtaining a novel bile acid sulfate sulfatase capable of specificallyhydrolyzing the sulfate ester moiety of bile acid 3α-sulfates, anddiscovered that total bile acid 3α-sulfates in blood or urine whichcould not be measured by the conventional enzymatic method can be easilydetermined by coupling said enzyme and β-hydroxysteroid dehydrogenase inthe presence of β-NAD, and also found that total bile acids includingthe bile acid 3α-sulfates in blood or urine could be easily determinedby coupling said enzyme, β-hydroxysteroid dehydrogenase and3α-hydroxysteroid dehydrogenase in the presence of β-NAD, and therebycompleted the invention.

The bile acid sulfate sulfatase of the invention (hereinafter referredto as the present Enzyme) is an enzyme produced by Pseudomonastestosteroni.

The physicochemical properties of the present Enzyme are furtherdescribed below. The activity of the present Enzyme is measured asfollows. That is, into a quartz cell are placed 0.1 ml of 2.5 mM aqueoussolution of lithocholic acid 3α-sulfate (Sigma), 0.2 ml of 15 mM 25aqueous solution of β-NAD (Oriental Yeast), 1.0 ml of 0.1 Mtris-hydrochloric acid buffer solution (pH 8.0) and 1.55 ml of distilledwater, and after equilibrating at 30° C., 0.05 ml of β-hydroxysteroiddehydrogenase (Sigma) solution (10 U/ml) and 0.1 ml of a solution of thepresent Enzyme are sequentially added thereto and the reaction iseffected at 30° C., and the increase of the absorbance at 340 nm in theinitial stage of the reaction is determined. Under these conditions, theenzyme activity producing 1 μmol of NADH per minute is defined as 1unit.

(a) Action: Acting on 3α-sulfates of bile acids to hydrolyze the sulfateester moiety and to invert the bonding configuration of the resulting OHgroup from α-configuration to β-configuration, thereby producing3β-hydroxy bile acids.

(b) Substrate specificity: Acting on 3α-sulfates of nonconjugated bileacids, and 3α-sulfates each of glycine-conjugated and taurine-conjugatedbile acids. The details are shown in Table 1.

                  TABLE 1                                                         ______________________________________                                                               Relative                                                                      activity                                               Substrate              (%)                                                    ______________________________________                                        Lithocholic acid 3α-sulfate                                                                    100                                                    Glycolithocholic acid 3α-sulfate                                                               11                                                     Taurolithocholic acid 3α-sulfate                                                               2                                                      Cholic acid 3α-sulfate                                                                         238                                                    3β-Hydroxy-5-cholenic acid 3-sulfate                                                            0                                                      4-Nitrocatechol sulfate                                                                              0                                                      p-Nitrophenyl sulfate  0                                                      ______________________________________                                    

(c) Optimum pH: pH 8.5±0.5 (FIG. 1). FIG. 1 is a graph showing therelation of the realtive activity of the present Enzyme and pH.

(d) pH stability: pH 5.6-7.6 (FIG. 2). FIG. 2 is a graph showing therelation of the realtive activity of the present Enzyme and pH when thepresent Enzyme is left to stand at 30° C. for 16 hours.

(e) Optimum temperature: 35° C. ±5° C. (FIG. 3). FIG. 3 is a graphshowing the relation between the realtive activity of the present Enzymeand temperature.

(f) Thermal stability: When the present Enzyme is left to stand at pH7.2 for 10 minutes, the activity remains by 100% at a temperature of 32°C. or lower, but at higher temperatures the activity rapidly decreasesand becomes 0% at 50° C. (FIG. 4). FIG. 4 is a graph showing the thermalstability of the present Enzyme.

(g) Molecular weight: As a result of measurement by high performanceliquid chromatography using gel filtration column Shim-pack Diol-300(Shimadzu), the molecular weight is calculated to be about 100,000.

(h) Km value: The Km value to lithocholic acid 3α-sulfate: 6×10⁻⁶ M.

(i) Inhibition and activation: Activated by Mn⁺⁺ and inhibited by thesubstances acting on metal ions, such as EDTA and o-phenanthroline, etc.Hardly inhibited by SH reagents such as p-chloromercuribenzoate andmonoiodoacetic acid. Details are shown in Table 2.

                  TABLE 2                                                         ______________________________________                                                                      Relative                                                          Concentration                                                                             Activity                                        Additives         (mM)        (%)                                             ______________________________________                                        No additive (control)                                                                           --          100                                             Magnesium sulfate 2           101                                             Manganese chloride                                                                              2           111                                             Cobalt chloride   2           92                                              Copper sulfate    2           94                                              Zinc sulfate      2           95                                              Cadmium chloride  2           98                                              Mercuric chloride 1           90                                              Mercuric chloride 2           71                                              EDTA              1           38                                              EDTA              5           25                                              o-Phenanthroline  1           37                                              α,α'-Dipyridyl                                                                      1           97                                              p-Chloromercuribenzoate                                                                           0.2       98                                              Monoiodoacetic acid                                                                             2           98                                              Sodium fluoride   2           98                                              Sodium azide      10          100                                             ______________________________________                                    

The present Enzyme is collected from the culture of Pseudomonastestosteroni. The Pseudomonas testosteroni (hereinafter called thisorganism) is not particularly limited, and any known strain may be used.Above all, Pseudomonas testosteroni ATCCll996 or the like is preferable.

The culture medium for use in the cultivation of this organism is notparticularly limited, and any culture medium may be used as far as thebacteria of the genus Pseudomonas can grow therein, and examples thereofinclude those containing yeast extract, peptone, meat extract or thelike as the organic nutrient sources, ammonium phosphate, ammoniumnitrate, potassium phosphate, sodium phosphate, magnesium chloride andmanganese chloride as the inorganic nutrient sources. Furthermore, toproduce the present Enzyme, it is essential to add a bile acid to theculture medium. Usable bile acids include, for example, cholic acid,deoxycholic acid, chenodeoxycholic acid, lithocholic acid sulfate andtheir salts, and these can be used singly or at least two of them may beused in combination. The amount of bile acid to be used is notparticularly limited, but is usually about 0.01 to 2.0 wt.%, preferablyabout 0.05 to 1.0 wt.%.

The cultivation is preferably done in aerobic condition. The cultivationtime and temperature are not particularly limited, but the cultivationtemperature is usually about 22° to 35° C., preferably about 26° to 30°C., and the cultivation time is usually about 8 to 30 hours, preferablyabout 12 to 24 hours. By cultivation for about 12 to 24 hours, theenzyme activity reaches its maximum.

From the bacterial cells harvested by the cultivation, the presentEnzyme can be obtained by, for example, extraction. The extraction isdone according to the conventional extraction methods of enzymes inbacterial cells. For example, the bacterial cells are destroyed byultrasonication, various mechanical processing or enzyme treatment, andthen centrifuged to separate the insoluble matter, whereby a supernatantcontaining the present Enzyme (crude enzyme solution) is obtained. Bypurifying this crude enzyme solution by properly selecting and combiningcommonly employed enzyme purification methods such as the treatment toremove nucleic acids, ammonium sulfate salting-out, ion exchangechromatography, hydrophobic chromatography and gel filtration, thepresent Enzyme can be isolated.

The present Enzyme thus obtained and β-hydroxysteroid dehydrogenase arecaused to act on a sample containing bile acid 3α-sulfates in thepresence of β-NAD, and thereby the bile acid 3α-sulfates are determined.That is, the present Enzyme converts the bile acid 3α-sulfates into3β-hydroxy bile acids, and then the β-hydroxysteroid dehydrogenaseconverts the 3β-hydroxy bile acids into 3-oxobile acids and concurrentlyreduces the NAD which is the coenzyme of said dehydrogenase into NADH.Therefore, when the conversion quantity from NAD into NADH isdetermined, the bile acid 3α-sulfates are determined.

The β-hydroxysteroid dehydrogenase is not particularly limited, and anyknown ones may be used.

The amounts of the enzymes to be used are not particularly limited, butusually the present Enzyme is used in an amount of about 0.04 to 2.0units, preferably 0.1 to 1.0 unit, and the β-hydroxysteroiddehydrogenase is usually used in an amount of about 0.05 to 2.0 units,preferably 0.1 to 1.0 unit. The amount of β-NAD to be used is also notparticularly limited, but it may be added such that the β-NADconcentration in the reaction system is usually about 0.1 to 10 mM,preferably about 0.5 to 5 mM. The enzyme reaction is usually conductedat a temperature of about 20° to 40° C., preferably about 25° to 37° C.,and for about 5 to 60 minutes, preferably about 10 to 30 minutes.

The NADH may be determined by a known method such as UV (340 nm)absorption measurement, fluorescence intensity measurement, reductivecoloring colorimetry, or calorimetric method wherein NADH oxidase iscaused to act on NADH to quantitatively generate from the NADH hydrogenperoxide which in turn is calorimetrically determined by oxidativecoloration.

Furthermore, in this invention, total bile acids including bile acid3α-sulfate can be determined by causing 3α-hydroxysteroid dehydrogenase,in addition to the present Enzyme and β-hydroxysteroid dehydrogenase, toact in the presence of β-NAD on a sample containing bile acid3α-sulfates and other bile acid(s). When urine or blood is used as thesample containing bile acid 3α-sulfates and other bile acid(s), thetotal bile acids in blood or urine are determined.

The 3α-hydroxysteroid dehydrogenase is not particularly limited, and anyknown ones may be used. The amount of the dehydrogenase to be used isnot particularly limited, but it is usually about 0.04 to 2.0 units,preferably about 0.1 to 1.0 unit. The amounts of other enzymes (thepresent Enzyme and β-hydroxysteroid dehydrogenase) and β-NAD, reactionconditions, and method of determination of the produced NADH may be thesame as in the case of determination of bile acid 3α-sulfates.

EXAMPLE

Examples are given below, but it must be noted that the invention is notlimited to these examples. In the examples, "%" means "wt.%".

EXAMPLE 1

A 300 ml quantity of a culture medium (pH 6.9) comprising 0.1% ammoniumdihydrogen phosphate, 0.1% diammonium hydrogen phosphate, 0.2% potassiumdihydrogen phosphate, 0.01% magnesium chloride, 0.01% manganese chlorideand 1.0% yeast extract was placed in a 2-liter Erlenmeyer flask, and wassterilized in an autoclave. Pseudomonas testosteroni ATCCll996 wasinoculated, and was cultivated for 24 hours at 28° C. under shaking.This seed culture broth was inoculated on 30 liters of sterilizedculture medium of the same composition as above in a 50-liter jarfermenter, and at the same time 1.2 liters of 10% sodium cholate aqueoussolution separately sterilized was aseptically added thereto, and thecultivation was continued for 15 hours at 28° C. under aeration andstirring.

The culture broth was centrifuged, and the obtained bacterial cells weresuspended in 30 mM phosphate buffer solution (pH 7.2), and thissuspension was applied to a DYNO-Laboratory mill (Willy A. BachofenMashinefabrik) to disrupt the cells. By centrifugation, the resultingsediments were removed, and crude enzyme solution was obtained. Thecrude enzyme solution was treated with protamine sulfate to removenucleic acid, followed by salting-out with use of ammonium sulfate. Thefraction precipitated under ammonium sulfate fractionation of 35 to 70 %saturation was collected, and dialyzed against 10 mM phosphate buffersolution (pH 7.2). The dialysate was passed through a column of DEAEcellulose (Whatman) equilibrated with 10 mM phosphate buffer solution(pH 7.2). The obtained non-adsorbed fractions were passed through acolumn of DEAE Sepharose CL-6B (Pharmacia) equilibrated with 5 mMtris-hydrochloric acid buffer solution (pH 8.0). The obtainednon-adsorbed fractions were concentrated by salting-out treatment withuse of ammonium sulfate, and allowed to be adsorbed on octyl SepharoseCL-4B (Pharmacia) column. Through this column 50 mM phosphate buffersolution was passed, and the eluting active fractions were concentratedby ultrafiltration, followed by gel filtration by passing through acolumn of Sephacryl S-200 (Pharmacia) equilibrated with 50 mM phosphatebuffer solution containing 0.15 M sodium chloride. The active fractionswere desalted and concentrated by ultrafiltration, thereby giving 480units of bile acid sulfate sulfatase. The purified product gave a singleband in slab gel electrophoresis with use of 7.5% acrylamide (pH 8.9).

EXAMPLE 2

Using the bile acid sulfate sulfatase obtained in Example 1, bile acid3α-sulfate was determined.

Each of aqueous solutions (0.1 ml each) of lithocholic acid 3α-sulfate,sodium salt (Sigma) or glycolithocholic acid 3α-sulfate, sodium salt(Sigma) having different concentrations was added, as substrate, to 2.9ml of 35 mM tris-hydrochloric acid buffer solution (pH 8.0) containing 3μmol of β-NAD (Oriental Yeast), 0.5 unit of β-hydroxysteroiddehydrogenase (Sigma) and 0.2 unit of bile acid sulfate sulfatase, andthe mixture was allowed to react for 10 minutes at 30° C., and then theabsorbance at 340 nm was measured. The results are shown in FIG. 5, fromwhich it is seen that the concentration of the substrate and theabsorbance are in positive correlation, thereby showing that the bileacid 3α-sulfates can be determined.

EXAMPLE 3

Reagent (1): 25.2 mg of β-NAD (Oriental Yeast), 7.6 mg of nitro-bluetetrazolium (Dojin Kagaku Kenkyusho), and 10 units of diaphorase (Sigma)were dissolved in 25 ml of 50 mM tris-hydrochloric acid buffer solution(pH 8.0) containing 0.3% NOIGEN ET-189 (nonionic surface active agent,made by Daiichi Kogyo Seiyaku).

Reagent (2): 0.4N hydrochloric acid

Measurement: A 1.25 ml quantity of Reagent (1) was added to each ofaqueous solutions (0.35 ml each) of lithocholic acid 3α-sulfate, sodiumsalt (Sigma) having various concentrations, and maintained at 37° C. for5 minutes. To this mixture were added 0.05 ml of β-hydroxysteroiddehydrogenase (Sigma) solution (10 units/ml) and 0.1 ml of bile acidsulfate sulfatase (2 units/ml) in the order mentioned and mixedtogether. The resulting mixture was maintained at 37° C. for 10 minutesto develop color. Then 1.25 ml of Reagent (2) was added to stop thereaction, and the absorbance at 560 nm was measured. Excellent resultsas shown in FIG. 6 were obtained.

EXAMPLE 4

The serum with addition of glycolithocholic acid 3α-sulfate (GLCA-S) wasdiluted with additive-free serum to various concentrations, therebygiving test sera.

To 0.2 ml of the test serum were added 0.15 ml of distilled water and1.25 ml of Reagent (1) used in Example 3, and the GLCA-S was determinedin the same manner as in Example 3. The results are shown in Table 3.

                  TABLE 3                                                         ______________________________________                                        GLCA-S concentration                                                          (μmol/liter)   Absorbance                                                  ______________________________________                                         50               0.045                                                       100               0.090                                                       150               0.137                                                       200               0.182                                                       ______________________________________                                    

EXAMPLE 5

The serum with addition of GLCA-S and glycolithocholic acid (GLCA) wasdiluted to various concentrations with additive-free serum, therebygiving test sera.

To 0.2 ml of test serum were added 0.15 ml of distilled water and 1.25ml of Reagent (1) of Example 3. Then the GLCA-S and GLCA were determinedin the same manner as in Example 3 except that a mixture ofβ-hydroxysteroid dehydrogenase and 3β-hydroxysteroid dehydrogenase(Sigma) (10 units/ml each) was used in place of β-hydroxysteroiddehydrogenase solution The results are shown in Table 4.

                  TABLE 4                                                         ______________________________________                                        GLCA-S concentration                                                                        GLCA concentration                                              (μmol/liter)                                                                             (←)         Absorbance                                     ______________________________________                                        25            25               0.044                                          50            50               0.091                                          75            75               0.135                                          100           100              0.180                                          ______________________________________                                    

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph showing the optimum pH of the present Enzyme.

FIG. 2 is a graph showing the pH stability of the present Enzyme.

FIG. 3 is a graph showing the optimum temperature of the present Enzyme.

FIG. 4 is a graph showing the thermal stability of the present Enzyme.

FIG. 5 and FIG. 6 are graphs showing the relation between theconcentration of the substrate (bile acid 3α-sulfate) and absorbanceobtained by practicing the method of the invention.

We claim:
 1. A biologically pure form of a bile acid sulfate sulfatasehaving the following properties:(a) action: acting on 3α-sulfates of5β-bile acids to hydrolyze the sulfate ester moiety thereof and tochange the bonding configuration of the resulting OH group fromα-configuration to β-configuration, thereby producting 3β-hydroxy bileacids; (b) substrate specificity: acting on 3α-sulfates ofnon-conjugated bile acids, and on 3α-sulfates each of gylcine-conjugatedand taurine-conjugated bile acids; (c) optimum pH range: pH 8.5±0.5; (d)optimum temperature range: 35°±5° C.; and (e) molecular weight asdetermined by high performance liquid chromatography using gelfiltration column: about 100,000.